Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4

Summary The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.     

Implication from the predicted docked interaction of sigma H and exploration of its interaction with RNA polymerase in Mycobacterium tuberculosis

M. tuberculosis is adapted to remain active in the extreme environmental condition due to the presence of atypical sigma factorscommonly called extra cytoplasmic function (ECF) sigma factors. Among the 13 sigma factors of M. tuberculosis, 10 are regarded asthe ECF sigma factor that exerts their attributes in various stress response. Therefore it is of interest to describe the structuralprediction of one of the ECF sigma factors, sigma H (SigH), involved in oxidative and heat stress having interaction with the β׳subunit of M. tuberculosis. RNA polymerase (Mtb-RNAP). The model of Mtb-SigH was build using the commercial package ofDiscovery Studio version 2.5 from Accelerys (San Diego, CA, USA) containing the inbuilt MODELER module and that of β׳ subunitof Mtb-RNAP using Phyre Server. Further, the protein models were docked using the fully automated web tool ClusPro(cluspro.bu.edu/login.php). Mtb-SigH is a triple helical structure having a putative DNA-binding site and the β׳ subunit of MtbRNAPconsists of 18-beta sheets and 22 helices. The SigH-Mtb-RNAP β׳ interaction studies showed that Arg26, Gln19 andAsp18,residues of SigH protein are involved in binding with Arg137, Gln140, Arg152, Asn133 and Asp144 of β׳ subunit of Mtb-RNAP.The predicted model helps to explore the molecular mechanism in the control of gene regulation with a novel unique target forpotential new generation inhibitor.